Method of selecting hla-dp4 ligands and the applications thereof

ABSTRACT

A method of selecting a test molecule that binds to HLA-DP4 by (i) incubating HLA-DP4 with the test molecule and a labeled peptide of formula (I) Z 1 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 Z 2  thereby forming respective complexes, wherein Z 1  and Z 2 , which may be identical or different, are each either zero or from 1 to 100 amino acids; X 1  is an aromatic or hydrophobic amino acid, or S; X 6  is an aromatic or hydrophobic amino acid, or C; X 9  is an aromatic or hydrophobic amino acid, or C, D, Q, S, T, or E; and X 2 , X 3 , X 4 , X 5 , X 7  and X 8  are each an amino acid; (ii) separating the respective complexes formed; (iii) detecting the HLA-DP4/labeled peptide complexes; and (iv) selecting the test molecule that exhibits a binding activity of IC 50 &lt;1000 nM, which corresponds to the concentration of the test molecule that inhibits 50% of the competitive HLA-DP4 binding of the labeled peptide.

The present invention relates to a method of selecting HLA-DP4 ligands and to the applications thereof.

CD4+ T lymphocytes are among the main cells which regulate the immune response. They are antigen-specific and are in fact capable of recognizing the presence of a pathogenic agent, of an allergen or of a tumor cell, and of triggering an immune response. Recognition of these antigens in fact results in the activation of the CD4+ T lymphocytes, which secrete most of the cytokines necessary for the recruitment of effector cells, namely cytotoxic CD8+ lymphocytes and antibody-producing B lymphocytes. CD4+ T lymphocytes are also involved in the activation of cells by cell contacts and, for example induce the activation, via the CD40 molecule, of antigen-presenting dendritic cells. CD4+ T lymphocyte activation is a favorable prognostic in infections with viruses such as the human immunodeficiency virus (HIV), human papilloma viruses (HPVs) or the hepatitis C virus (HCV), and these cells appear to be necessary for antitumor immunity. Their role is not, however, systematically beneficial for the organism. Autoimmune diseases very commonly result from uncontrolled activation of CD4+ T lymphocytes. This is the case of multiple sclerosis and of insulin-dependent diabetes. These lymphocytes also participate in the establishment of allergic diseases. IL4, which is mainly secreted by CD4+ T lymphocytes, is in fact the main factor which results in the production of IgE. Finally, the role of CD4+ T lymphocytes in the triggering of transplant rejection is well established. Thus, depending on the disease, treatments are aimed at triggering the activation of CD4+ T lymphocytes (immunization against a pathogenic agent or a tumor cell) or at decreasing the state of activation of CD4+T lymphocytes (desensibilization against the allergy, prevention of transplant rejection).

The activation of CD4+ T lymphocytes takes place under the effect of the presentation of antigenic peptides by the molecules of the major histocompatibility complex type II borne by the antigen-presenting cells (APCs); in humans, they are called HLA II molecules for human leukocyte Antigen type II. These antigenic peptides, called T epitopes, result from the proteolytic degradation of the antigens by the antigen-presenting cells. They are variable in length, generally from 13 to 25 amino acids, and have a sequence which makes them capable of binding to the HLA II molecules. It is well known that, just like the native antigen, a T-epitope peptide is capable of stimulating, in vitro, CD4+ T lymphocytes which are specific for them, or of recruiting them in vivo. It is therefore sufficient to induce a CD4+ T response. Interestingly, it is also known that, according to the modes of presentation (route of administration, doses, possible addition of an adjuvant), the recognition of these peptides leads either to activation of the CD4+ T lymphocytes (ALEXANDER et al., J. Immunol., 2000, 164, 1625-1633; DEL GUERCIO et al., Vaccine, 1997, 15, 441-448; FRANKE et al., Vaccine, 1999, 17, 1201-1205), or to anergy thereof (MULLER et al., J. Allergy Clin. Immunol., 1998, 101, 747-754; OLDFIELD et al., J. Immunol., 2001, 167, 1734-1739). These T-epitope peptides are therefore capable both of contributing to the composition of a vaccine or of serving to decrease the undesired activation of CD4+ T lymphocytes. They are also capable of contributing to a test for diagnosing the immune state of patients or of normal individuals, based on the direct detection (lymphocyte proliferation assay) or indirect detection (production of antibodies, of cytokines, etc.) of said activated CD4+ T lymphocytes. One of the major problems which limits the use of these peptides is the identification of these epitopes, given that their sequence varies from one individual to another due to the polymorphism of the HLA II molecules. In fact, the HLA II molecules are hetero dimers consisting of an alpha (α)-chain and of a beta (β)-chain which are polymorphic. Four types of HLA II molecules exist per individual (2 HLA-DR, 1 HLA-DQ and 1 HLA-DP). The HLA-DR molecule, the beta (β)-chain of which is encoded by the DRB1 gene, is the most commonly expressed. To date, the betα-chain encoded by the DRB1 gene is the most polymorphic and has 273 alleles. For the HLA-DQ and HLA-DP molecules, the two chains (α and β) of which they are formed are polymorphic but they have fewer alleles. There are in fact 21 DQA1 alleles (α-chain of HLA-DQ), 45 DQB1 alleles (β-chain of HLA-DQ), 19 DPA1 alleles (α-chain of HLA-DP) and 93 DPB1 alleles (β-chain of HLA-DP). However, combination between the two α- and β-chains encoded by these alleles gives rise to many HLA-DQ and HLA-DP molecules. Because of this polymorphism, these isoforms possess binding properties which are different from one another, which implies that they can bind different peptides of the same antigen. Thus, each individual recognizes in an antigen a set of peptides, the nature of which depends on the HLA II molecules which characterizes said individual. Since a large number of HLA II alleles exists, it may be assumed that there exists, in a given sequence, a considerable repertoire of T-epitope peptides having very different sequences, each one specific for a different allele.

However, this HLA II molecule diversity is not as great on the scale of each population as on a worldwide scale, as illustrated by table I below.

TABLE I Gene frequencies for the HLA class II molecules that are most abundant in the Caucasian population (Europe, USA)*, according to J. Colombani, 1993, HLA: immune functions and medical applications, published by John Libbey Eurotext. Molecules α-chain Frequency (%) β-chain Frequency (%) Abundant molecules HLA-DR DRA*0101 100 DRB1*0101 9.3 DRA*0101/DRB1*0101 DRB1*1501 8.0 DRA*0101/DRB1*1501 DRB1*0301 10.9 DRA*0101/DRB1*0301 DRB1*0401 5.6 DRA*0101/DRB1*0401 DRB1*1101 9.2 DRA*0101/DRB1*1101 DRB1*1301 6.0 DRA*0101/DRB1*1301 DRB1*0701 14.0 DRA*0101/DRB1*0701 DRB3*0101 9.2 DRA*0101/DRB3*0101 DRB3*0202 12.0 DRA*0101/DRB1*0202 DRB4*0101 28.4 DRA*0101/DRB4*0101 DRB5*0101 7.9 DRA*0101/DRB5*0101 HLA-DQ DQA1*0101 17.0 DQB1*0501 14.9 DQA1*0101/DQB1*0501 DQA1*0102 15.8 DQB1*0602 9.8 DQA1*0501/DQB1*0301 DQA1*0201 12.4 DQB1*0603 5.8 DQA1*0501/DQB1*0201 DQA1*0301 14.5 DQB1*0201 21.3 DQA1*0301/DQB1*0302 DQA1*0501 20.9 DQB1*0301 12.0 DQA1*0102/DQB1*0602 DQB1*0302 13.0 DQA1*0201/DQB1*0201 HLA-DP DPA1*0103 78.2 DPB1*0101 7.1 DPA1*0201/DPB1*0101 DPA1*0201 21.2 DPB1*0201 11.9 DPA1*0103/DPB1*0201 DPB1*0301 9.1 DPA1*0103/DPB1*0301 DPB1*0401 40.1 DPA1*0103/DPB1*0401 DPB1*0402 11.0 DPA1*0103/DPB1*0402 *The gene frequencies for the 2 HLA-DP4 molecules are indicated in bold.

Thus, for the HLA-DR and HLA-DQ molecules, about ten alleles are sufficient to cover more than 60% of the gene frequency found in the Caucasian population and therefore to involve more than 85% of the Caucasian population.

For the HLA-DP molecules, the most common molecule is the DP4 molecule derived from the DPA1*0103 and DPB1*0401 alleles, which have gene frequencies of 78.2% and 40%, respectively. Three other DP molecules have frequencies which exceed 5%: a DP3 molecule (DPA1*0103/DPB1*0301), a DP2 molecule (DPA1*0103/DPB1*0201) and a DP4 molecule (DPA1*0103/DPB1*0402); the HLA II molecules are named as a function of the DPB1 allele encoding the β-chain which is the most polymorphic.

Thus, four HLA-DP molecules (1 DP3 molecule, 1 DP2 molecule and 2 DP4 molecules) are therefore sufficient to cover 71% of the gene frequency of the Caucasian population, the two DP4 molecules covering 51% by themselves. Each of these DP4 molecules comprises a variable α-chain encoded either by DPA1*0103, which is the most common (78.2%), or by DPA1*0201 (20.2%), the two α-chains differing only at the level of 3 amino acids (positions 31, 50 and 83; table II). The HLA-DP4 molecules which exhibit a high allelic frequency in the Caucasian population (of the order of 50% in Europe and 80% in North America) are also present at not insignificant frequencies in the other populations (allelic frequency of the order of 60% in South America, 60% in India, 25% in Africa and 15% in Japan, Colombani et al., mentioned above).

TABLE II Polymorphism of the α- and β-chains of the HLA-DP molecules in the Caucasian population (Europe, USA), according to J. Colombani, mentioned above. Polymorphic positions* Alleles Freq (%) 8 9 11 37 38 57 58 59 67 71 78 86 87 88 89 β-chain 401 40.1 L F G F A A A E I K M G G P M 402 11.0 — — — — V D E — — — — — — — — 201 11.9 — — — — V D E — — E — — — — — 501 1.3 — — — L V E — — — — — D — — — 101 7.1 V Y G Y — — — — — — — D E A V 301 9.1 V Y L — V D E D L — V D E A V 901 1.1 V H L — V D E D I E V D E A V Alleles Freq (%) 31 50 83 α-chain 103 78.2 M Q T 201 21.2 Q R A *The sequences, available on the internet site http://www.ebi.ac.uk/imgt/hla, are numbered according to the numbering of STERN et al. (Nature, 1994, 368, 215-221) for the HLA-DR molecule; as the residues at positions 23 and 24 are absent in the DPB sequences, the residues indicated at positions 37, 38, 57, 59, 67, 71, 78, 86, 87, 88 and 89 correspond respectively to positions 35, 36, 55, 56, 57, 65, 69, 76, 84, 85, 86 and 87 in the DPB sequence.

However, despite the high frequencies, each of these DP4 molecules has only been very partially studied; the most common studies in fact result from the isolation of HLA-DP4 molecule-restricted CD4+ T lymphocyte clones specific for the peptides given in table III below, derived from the following antigens:

-   -   Antigens from pathogenic microorganisms, such as the tetanus         toxin (WYSS CORAY et al., Eur J. Immunol., 1992, 22, 2295), the         Blastomyces dermatitidis WI-1 antigen (CHANG et al., Inf.         Immun., 2000, 68, 502), the Mycobacterium Bovis hsp 65 protein         (GASTON et al., Int. Immunol., 1991, 3, 965-972), the hepatitis         B virus S antigen (HBsAg; CELIS et al., J. Virol., 1989, 63,         747), the rabies virus non-structural phosphoprotein (RV-NS) or         influenza B virus neuraminidase (IBV-Nm; CELIS et al., J.         Immunol., 1990, 145, 305) and the herpes simplex virus type 1         UL21 protein (KOELLE et al., J. Virol., 2000, 74, 10930-10938),     -   allergens such as the major allergen of Dermatophagoides         pteronyssinus (HIGGINS, J. Allergy Clin. Immunol., 1992, 90,         749), and     -   tumor antigens such as MAGE-A3 (SCHULTZ et al., Cancer Res.,         2000, 60, 6272) and NY-ESO1 (ZENG et al., P.N.A.S., 2001, 98,         3964-3969).

TABLE III Sequences of the peptides which are ligands for the HLA-DP4 molecules Peptides Sequences Identification number Reference TT947-967 FNNFTVSFWLRVPKVSASHLE SEQ ID NO: 1 Wyss Coray et al., 1992 PSN 265 DPYNCDWDPYHEKYDWDLWNK SEQ ID NO: 2 Chang et al., 2000 WCN Sld (HBsAg 19-28) FFLLTRILTI SEQ ID NO: 3 Celis et al., 1989 NS-p2 (RV NS 101-120) GVQIVRQIRSGERFLKIWSQ SEQ ID NO: 4 Celis et al. 1990 FLU-p1 (IBV Nm 247-260) GISKCRFLKIREGR SEQ ID NO: 5 Celis et al., 1990 MT451-466 IAFNSGMEPGVVAEKV SEQ ID NO: 6 Gaston et al., 1991 MT456-471 GMEPGVVAEKVRNLSV SEQ ID NO: 7 Gaston et al., 1991 Derp 101-119 PNAQRFGISNYCQIYP SEQ ID NO: 8 Higgins, 1992 MAG 247-258 TQHFVQENYLEY SEQ ID NO: 9 Schultz et al., 2000 NY-ESO1 161-180 MWITQCFLPVFLAQPPSGQR SEQ ID NO: 10 Zeng et al., 2001 NY-ESO1 156-175 QLSLLMWITQCFLPVFLAQPP SEQ ID NO: 11 Zeng et al., 2001 UL21 283-293 RELWWVFYAGD SEQ ID NO: 12 Koelle et al., 2000 IL3 127-146 GPGAPADVQYDLYLNVANRR SEQ ID NO: 13 Falk et al., 1994 UNK1 (UNK 1-17) EKKYFAATQFEPLAARL SEQ ID NO: 14 Falk et al., 1994 UNK2 (UNK 2-17) KKYFAATQFEPLAARL SEQ ID NO: 15 Falk et al., 1994 UNK3 (UNK 1-13) EKKYFAATQFEPL SEQ ID NO: 16 Falk et al., 1994

The abovementioned studies which are based on the isolation of HLA-DP4 molecule-restricted CD4+ T lymphocyte clones use a functional assay (proliferation assay) which has not made it possible to demonstrate a DP4 molecule-binding unit shared by all these peptides. In addition, this assay is very laborious to implement due to the fact that it requires a large number of correctly sampled patients, so that they represent the diversity of HLA II molecules of the entire population. In addition, the epitopes defined are those used by the immune system of the patients during natural infection with an antigen; these epitopes are not necessarily the most effective for inducing an immune response against this same antigen.

Using another approach, namely the analysis of four peptides naturally present on DP4 molecules [peptide 127-146 of the α-chain of the IL3 receptor (IL3 127-146; SEQ ID NO: 13) and three peptides of unknown origin: UNK1 (SEQ ID NO:14), UNK2 (SEQ ID NO:15) and UNK3 (SEQ ID NO:16); table III], FALK et al. (Immunogenetics, 1994, 39, 230-242) have proposed a DP4 molecule-binding consensus sequence. This sequence comprises three anchoring residues, respectively at positions P1, P7 and P9/P10. P1 and P7 are hydrophobic or aromatic (Y, V, L, F, I, A, M, W) and P9/P10 are preferably aliphatic; however, a Y residue is tolerated at positions P9/P10, but not an F residue. The residues P1 and P7 are preceded by groups of charged residues (K, R, E, N, Q for P1 and N, K, E for P7), and small residues (A, V) are common at position P3 and position P9. Table III shows that the only other DP4-ligand peptides exhibiting this unit are the overlapping peptides NY-ESO1 161-180 and NY-ESO1 156-175. Consequently this unit proposed by FALK et al. does not make it possible to define a DP4 molecule-binding unit shared by all the peptides that are ligands for the DP4 molecules, identified by functional assays.

Although DP9 molecule-binding assays (DONG et al., J. Immunol. 1995, 154, 4536-4545) and DP2 molecule-binding assays (CHICZ et al., J. Immunol., 1997, 159, 4935-4942), derived from those developed from the DR molecules [MARSHALL et al., J. Immunol., 1994, 152, 4946 (HLA-DR1); patent FR 99 0879 and TEXIER et al., J. Immunol., 2000, 164, 3177 (HLA-DR1, -DR2, -DR3, -DR4, -DR7, -DR11 and -DR13)] make it possible to isolate peptides specific for, respectively, DP9 and DP2, these assays do not make it possible to isolate peptides specific for DP4 (CHICZ et al., mentioned above): the peptides isolated using the DP2-binding assay have a high affinity for DP2 (binding activity <10 nM), whereas peptides known to be DP4-restricted, such as the HBsAg 14-33 peptide, exhibit moderate activity (20 μM).

In addition, due to the considerable differences in the residues of the binding site, between the principal DP molecules, the binding assays developed for these molecules do not make it possible to identify DP4-restricted peptides.

Thus, the binding units shared by all the peptides capable of binding to HLA-DP4 molecules have not been identified, in particular due to the fact that there is no method of identifying such peptides which is simple to implement and suitable for the simultaneous screening of a large number of peptides, such as banks of overlapping peptides representing the antigen sequence of interest.

Nevertheless, given the frequency of the DP4 molecules, the peptides which bind to the DP4 molecules constitute candidate peptides for specific immunotherapy and immunization and could be used for diagnosing the immune state of patients or of normal individuals.

The inventors have therefore developed a method of selecting HLA-DP4 ligands, which has enabled them to isolate HLA-DP4-specific ligands, in particular peptides, and to specify the binding units shared by the HLA-DP4-ligand peptides, based on the peptides obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

Besides the above arrangements, the invention also comprises other arrangements, which will emerge from the following description which refers to examples of implementation of the subject of the present invention, with references to the attached drawings in which:

FIG. 1 illustrates the activity of binding of peptides to the HLA-DP4 molecules encoded, respectively, by DPB1*0401 (A) and DPB1*0402 (B), determined according to the method in accordance with the invention with, as tracer peptide, the biotinylated UNK1 peptide (10 nM); the percentage of DP4 molecule-binding is expressed as a function of the molar concentration of the peptides. The maximum binding (100%) corresponds to the value obtained for the tracer peptide alone, in the absence of competitor peptide. UNK: UNK1 (SEQ ID NO:14), IL: IL3 127-146 (SEQ ID NO:13), MAG: MAG 245-258 (SEQ ID NO:19), NSP2: SEQ ID NO:4, TT: TT 947-963 (SEQ ID NO:20), DQB: DQB 43-57 (SEQ ID NO:23), HCI 46-63: SEQ ID NO:24) and HA: HA 306-318 (SEQ ID NO:21),

FIG. 2 illustrates the correlation between the HLA-DP4-binding score (estimated by the method of identifying HLA-DP4-ligand peptides in accordance with the invention) and the affinity for the HLA-DP4 molecules (determined by the IC₅₀ value, measured using the HLA-DP4-binding assay defined in the method of selecting HLA-DP4-ligand peptides in accordance with the invention) analyzed on a set of 44 peptides.

Consequently, a subject of the present invention is a method of selecting HLA-DP4-ligand molecules, comprising the following steps:

-   -   (i) incubation of purified HLA-DP4 with a tracer consisting of a         prelabeled peptide and able to be detected by an appropriate         signal, which tracer peptide is chosen from the group consisting         of the peptides exhibiting a signal/background noise ratio         greater than 5, at a concentration of 10 nM, in a direct         HLA-DP4-binding assay, and corresponding to general formula (I)         Z₁X₁X₂X₃X₄X₅X₆X₇X₈X₉Z₂ in which:         -   Z₁ and Z₂, which may be identical or different, are zero or             each represent a peptide of 1 to 100 natural or synthetic             amino acids, preferably of 1 to 30 amino acids, even more             preferably of 1 to 10 amino acids;         -   X₆ represents an aromatic or hydrophobic amino acid, or a             cysteine (C);         -   X₁ represents an aromatic or hydrophobic amino acid and/or             X₉ represents an aromatic or hydrophobic amino acid, or a             cysteine (C), an aspartic acid (D), a glutamine (Q), a             serine (S), a threonine (T) or a glutamic acid (E); and         -   X₂, X₃, X₄, X₅, X₇ and X₈ each represent a natural or             synthetic amino acid, in the presence of various             concentrations of test molecule(s);     -   (ii) separation of the various complexes formed;     -   (iii) detection of the HLA-DP4/tracer peptide complexes by         measuring the signal associated with said tracer peptide; and     -   (iv) selection of the ligand molecules which exhibit a binding         activity IC₅₀<1000 nM, corresponding to the concentration of         these molecules which inhibits 50% of the binding of the tracer         peptide.

The residues X₁, X₆ and X₉ of general formula (I) as defined above, which constitute the residues for anchoring in the pockets of the HLA-DP4 molecule, are also called, respectively, residues P1, P6 and P9. Among these residues, the residues X1 (or P1) and X6 (or P6) are the residues which provide the main contribution to the binding to HLA-DP4. The residue X₉ or P₉ is less important and provides less contribution to the binding to HLA-DP4.

For the purpose of the present invention:

-   -   the expression “natural or synthetic amino acids” is intended to         mean the 20 natural α-amino acids commonly found in proteins         (one-letter code: A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P,         S, T, W, Y and V), certain amino acids rarely encountered in         proteins (hydroxyproline, hydroxylysine, methyllysine,         dimethyl-lysine, etc.), amino acids which do not exist in         proteins, such as β-alanine, γ-aminobutyric acid, homocysteine,         ornithine, citrulline, canavanine, norleucine,         cyclohexylalanine, etc., and the enantiomers and         diastereoisomers of the above amino acids;     -   the term “hydrophobic amino acid” is intended to mean an amino         acid selected from (one-letter code): A, V, L, I, P, W, F and M;     -   the term “aromatic amino acid” is intended to mean an amino acid         selected from (one-letter code): F, W and Y.

The use of a tracer peptide, as defined in step (i), makes it possible to effectively select ligands specific for HLA-DP4, i.e. molecules, in particular peptides, which exhibit good affinity for HLA-DP4, i.e. a binding activity <1000 nM.

A tracer peptide in accordance with the invention is selected by carrying out a direct HLA-DP4-binding assay, for example by following steps (i), (ii) and (iii) of the protocol defined above, but in the absence of competitor, corresponding to the test molecule. The appropriate signal detected (fluorescence, etc.) reveals the HLA-DP4/tracer peptide complexes [step (iii)] and the background noise represents the corresponding signal, obtained in the absence of HLA-DP4.

Preferably:

-   -   X₆ is selected from L, I, W, F, M, Y and C, and

X₁ is selected from A, V, L, I, W, F, M and Y, and/or X₉ is selected from A, V, L, I, P, W, F, M, Y, C, D, Q, S, T and E.

Tracer peptides in accordance with the invention are represented by the peptides NS-p2 (SEQ ID NO:4), MAG 247-258 (SEQ ID NO:9), UL21 283-293 (SEQ ID NO:12), IL3 127-146 (SEQ ID NO:13), UNK1 (SEQ ID NO:14), UL21 283-302 (SEQ ID NO:18) and MAG 245-258 (SEQ ID NO:19).

According to an advantageous embodiment of said method, the tracer peptide is chosen from the group consisting of biotinylated peptides, radiolabeled peptides and peptides coupled to a fluorochrome.

In step (ii), the separation of the formed complexes from the unbound peptides is carried out, for example, by transfer of the formed complexes onto a microtitration plate precoated with an HLA-DP-specific antibody, by chromatography on a gel filtration column or by centrifugation.

When the tracer peptide is radiolabeled or coupled to a fluorochrome, in particular to europium, the HLA-DP4/tracer peptide complexes are detected directly by measuring the radioactivity or the fluorescence emitted by said complexes.

When the tracer peptide is biotinylated, the HLA-DP4/tracer peptide complexes are detected indirectly using conjugated streptavidin, for example by immuno-enzymatic detection using streptavidin conjugated to an enzyme such as alkaline phosphatase, and a substrate for alkaline phosphatase such as 4-methylumbelliferyl phosphate (MUP).

According to another advantageous embodiment of said method, the tracer peptide is used at a concentration <200 nM, preferably less than 20 nM, even more preferably the tracer is used at a concentration of 10 nM.

According to yet another advantageous embodiment of said method, said HLA-DP4 in step (i) is chosen from the group consisting of the molecules encoded by the DPA1*103/DPB1*0401 and DPA1*103/DPB1*0402 alleles.

The method according to the invention advantageously makes it possible to select any HLA-DP4 ligand; this involves both mineral or organic molecules such as peptides and pseudopeptides.

According to another advantageous embodiment of said method, said test molecules represent a library of overlapping peptides covering the sequence of an antigen.

A subject of the present invention is also HLA-DP4 ligands which can be obtained by the method of selection as defined above, corresponding to a mineral or organic, natural or synthetic molecule exhibiting an HLA-DP4-binding activity of less than 1000 nM.

The HLA-DP4-binding activity of a ligand molecule, as defined above, corresponds to the concentration of said ligand molecule which inhibits 50% of the HLA-DP4-binding of a labeled tracer peptide, in a competition assay such as the method of selecting HLA-DP4 ligands defined above.

Among these ligand molecules, mention may in particular be made of peptides and modified peptides such as glycopeptides, lipopeptides, and peptides comprising D-amino acids, pseudopeptide bonds (pseudopeptides) or modifications of the C- or N-terminal ends.

The lipid portion of the ligand lipopeptide is in particular obtained by addition of a lipid unit to an α-amino function of said peptides or to a reactive function of the side chain of an amino acid of the peptide portion; it may comprise one or more chains, derived from C₄-C₂₀ fatty acids, which are optionally branched or unsaturated (palmitic acid, oleic acid, linoleic acid, linolenic acid, 2-aminohexadecanoic acid, pimelautide, trimexautide) or derived from a steroid. The method of preparing such lipopeptides is in particular described in international applications WO 99/40113 and WO 99/51630. The preferred lipid portion is in particular represented by an N^(α)-acetyl-lysine N^(ε) (palmitoyl) group, also referred to as Ac-K (Pam).

According to an advantageous embodiment of said HLA-DP4-ligand peptide as defined above, its peptide sequence corresponds to general formula (I) Z₁X₁X₂X₃X₄X₅X₆X₇X₈X₉Z₂, in which:

-   -   Z₁ and Z₂, which may be identical or different, are zero or each         represent a peptide of 1 to 10 amino acids as defined above,         preferably of 1 to 30 amino acids, even more preferably of 1 to         100 amino acids;     -   X₆ represents an aromatic or hydrophobic amino acid, or a         cysteine (C);     -   X₁ represents an aromatic or hydrophobic amino acid and/or X₉         represents an aromatic or hydrophobic amino acid, or else a         cysteine (C), an aspartic acid (D), a glutamine (Q), a serine         (S), a threonine (T) or a glutamic acid (E); and     -   X₂, X₃, X₄, X₅, X₇ and X₈ each represent a natural or synthetic         amino acid, on condition that said HLA-DP4-ligand peptide of         general formula (I) does not correspond to any of the sequences         SEQ ID NOS: 1 to 17.

According to an advantageous embodiment of said ligand peptide:

-   -   X₆ is selected from L, I, W, F, M, Y and C, and     -   X₁ is selected from A, V, L, I, W, F, M and Y, and/or X₉ is         selected from A, V, L, I, P, W, F, M, Y, C, D, Q, S, T and E.

According to an advantageous arrangement of this embodiment, said ligand peptide binds specifically to DPB1*0401 (DPB1*0401-binding activity at least two times greater than the DPB1*0402-binding activity) and X₆ is different from C, and/or X₁ is different from A and from V, and/or X₉ represents W or Y or X₉ is different from E and C, and/or X₄ is different from K and R.

According to another advantageous arrangement of this embodiment, said ligand peptide binds specifically to DPB1*0402 (DPB1*0402-binding activity at least two times greater than the DPB1*0401-binding activity) and X₆ represents C, and/or X₁ represents A or V, and/or X₉ represents E or C or X₉ is different from Y and W, and/or X₄ represents K or R.

According to another advantageous embodiment of said ligand peptide, Z₁ and Z₂ are chosen from the group consisting of:

-   -   the sequences of the antigen which are adjacent to the         HLA-DP4-restricted CD4+ epitope as defined above, and/or     -   one or more CD8+ T epitopes, and/or     -   multiple CD4+ epitopes, such as peptide 830-846 of the tetanus         toxin TT (O′SULLIVAN et al., J. Immunol., 1991, 147, 2663-2669),         peptide 307-319 of influenza virus hemagglutinin HA (O′SULLIVAN         et al., mentioned above), the Pan DR or PADRE epitope (ALEXANDER         et al., DEL GUERCIO et al., FRANKE et al., mentioned above) and         peptides derived from the antigens of Plasmodium falciparum,         such as the CS.T3 peptide (SINIGAGLIA et al., Nature, 1988, 336,         778-780) and the CSP, SSP2, LSA-1 and EXP-1 peptides (DOOLAN et         al., J. Immunol., 2000, 165, 1123-1137) and/or     -   one or more B epitopes, for example a peptide or a glycopeptide         in which said B epitope consists of a sugar (ALEXANDER et al.,         mentioned above).

Such sequences advantageously make it possible to trigger or to modulate an immune response in an appropriate manner.

According to yet another advantageous embodiment of said ligand peptide, it has the sequence SEQ ID NO:84 corresponding to the NY-ESO1 87-111 peptide.

The present invention also encompasses the ligand peptides as defined above, which have been polymerized.

A subject of the present invention is also a method of identifying HLA-DP4-ligand peptides as defined above, based on an amino acid sequence, characterized in that it comprises at least the following steps:

-   -   a) establishing an HLA-DP4-binding matrix by calculating—for all         the mutants of a tracer peptide as defined above, representing         all the substitutions of the residues at position 1, 4, 6 or 9         of said tracer peptide with the other 19 natural amino acids—the         ratio of the IC₅₀ values for said mutant peptides and for said         tracer peptide, using the HLA-DP4-binding assay as defined in         the method above,     -   b) evaluating the HLA-DP4-binding of peptides of at least 9         amino acids included in said amino acid sequence, by         calculating, for each fragment of 9 amino acids of said peptide,         the sum of the scores for HLA-DP4-binding of the residues at         positions 1, 4, 6 and 9 of said fragment, using the binding         matrix established in a), and     -   c) identifying the HLA-DP4-ligand peptides corresponding to         those to which the loss of HLA-DP4-binding, relative to said         tracer peptide, is smallest, i.e. those for which the sum of the         binding scores has the lowest value expressed as a logarithm         (log); preferably less than 2, preferably less than 1, even more         preferably close to 0.

This HLA-DP4-binding matrix which is illustrated for the reference peptide UNK 3-15 (SEQ ID NO:28), in table XIV of example 6, makes it possible to estimate the HLA-DP4-binding activity of any peptide of at least 9 amino acids; peptides having binding scores of, respectively, 0, 1, 2, 3 and 4 correspond to peptides exhibiting a 1-fold, 10-fold, 100-fold, 1000-fold and 10 000-fold loss of binding relative to the UNK 3-15 peptide (IC₅₀ 10 nM), i.e. exhibiting an estimated binding activity of, respectively, 10 nM, 100 nM, 1000 nM, 10 μM and 100 μM.

For example, the tumor antigen NY-ESO1 comprises a peptide WITQCFLPV having a DP*0401- and DP*0402-binding score of 0+0.3+0+0.3=0.6 corresponding to an estimated binding activity (using the HLA-DP4-binding matrix) of 39 nM and a calculated activity (using the DP*0401- and DP*0402-binding assay) of, respectively, 20 nM and 67 nM.

The method of identifying HLA-DP4-ligand peptides according to the invention, which is easy to implement and can be automated, makes it possible, in particular using appropriate software, to predict the sequence of the HLA-DP4-ligand peptides present in all proteins representing antigens of interest. The peptide sequences thus identified can then be verified using an HLA-DP4-binding assay, defined in the method of selecting HLA-DP4 ligands according to the invention.

A subject of the present invention is also a nucleic acid molecule, characterized in that it encodes a ligand peptide as defined above.

The subject of the invention also encompasses the recombinant nucleic acid molecules comprising at least one nucleic acid molecule in accordance with the invention, linked to at least one heterologous sequence.

For the purpose of the present invention, the expression “sequence which is heterologous relative to a nucleic acid sequence encoding a ligand peptide” is intended to mean any nucleic acid sequence other than those which, naturally, are immediately adjacent to said nucleic acid sequence encoding a peptide.

The subject of the present invention encompasses in particular:

-   -   expression cassettes comprising at least one nucleic acid         molecule in accordance with the invention and the sequences         required for controlling the transcription and the translation         of said nucleic acid molecule (promoter, intron, initiation         codon (ATG), stop codon, polyadenylation signal), and     -   recombinant vectors comprising an insert consisting of a nucleic         acid molecule in accordance with the invention. Advantageously,         these expression vectors comprise at least one expression         cassette as defined above.

Many vectors into which a nucleic acid molecule of interest can be inserted in order to introduce it into and to maintain it in a eukaryotic or prokaryotic host cell are known in themselves; the choice of a suitable vector depends on the use envisioned for this vector (for example, replication of the sequence of interest, expression of this sequence, maintenance of this sequence in extrachromosomal form, or else integration into the host's chromosomal material), and also on the nature of the host cell.

For example, use may be made, inter alia, of viral vectors such as adenoviruses, retroviruses, lentiviruses and AAVs, into which the sequence of interest has been inserted beforehand; said sequence (isolated or inserted into a plasmid vector) can also be combined with a substance which allows it to cross the host cell membrane, for example a preparation of liposomes, of lipids or of cationic polymers, or it can be injected directly into the host cell, in the form of naked DNA.

A subject of the invention is also prokaryotic or eukaryotic cells transformed with at least one nucleic acid molecule in accordance with the invention.

Transformed cells in accordance with the invention can be obtained by any means, known in themselves, making it possible to introduce a nucleic acid molecule into a host cell. For example, in the case of animal cells, use may be made of the vectors or the lipid preparations as defined above.

A subject of the present invention is also an immunomodulatory composition, characterized in that it comprises at least one HLA-DP4 ligand or a nucleic acid molecule encoding an HLA-DP4-ligand peptide as defined above, and a pharmaceutically acceptable vehicle.

Advantageously, said nucleic acid molecule is included in a vector as defined above.

Depending on the choice of the HLA-DP4 ligand and of its mode of presentation (route of administration, dose, possible addition of adjuvant) such an immunomodulatory composition results in either activation of T lymphocytes, or the anergy thereof.

In fact, it has been shown that a single subcutaneous injection of a small amount of peptide results in anergy (OLDFIELD et al., J. Immunol., 2001, 167, 1734-1739). On the other hand, it is known that repeated injections of large amounts of peptide in the presence of adjuvant result in T lymphocyte activation. In addition, culmination with Z₁ and Z₂ peptides as defined above also makes it possible to increase the antigen-specific immune response (ALEXANDER et al., mentioned above).

Consequently, said composition is used both for immunization against a pathogenic agent or a tumor cell and for the treatment of autoimmune diseases (multiple sclerosis, insulin-dependent diabetes), of allergy or of transplant rejection.

A subject of the present invention is also a reagent for diagnosing the immune state of an individual, characterized in that it comprises at least one HLA-DP4 ligand as defined above, optionally labeled or complexed, in particular complexed with labeled (biotinylated) HLA-DP4 molecules, in the form of multimeric complexes such as tetramers.

A subject of the present invention is also the use of an HLA-DP4 ligand or of a nucleic acid molecule encoding an HLA-DP4-ligand peptide as defined above, for preparing an immunomodulatory medicinal product or a reagent for diagnosing the immune state of an individual.

For the purpose of the present invention, the expression “diagnosing the immune state of an individual” is intended to mean detecting the presence, in said individual, of CD4+ T lymphocytes specific for an antigen derived from a pathogenic agent or from a tumor cell, for an allergen, for an alloantigen or for an autoantigen.

The reagent in accordance with the invention which is capable of detecting the presence of CD4+ T lymphocytes specific for an antigen is used for detecting: an infection with a pathogenic agent, a cancer, an autoimmune disease, an allergy or a transplant rejection, based on a biological sample from a patient.

A subject of the present invention is also a method of diagnosing the immune state of an individual, comprising the steps of:

-   -   bringing a biological sample from said individual into contact         with a diagnostic reagent as defined above, and     -   detecting the CD4+ T lymphocytes specific for an antigen by any         suitable means.

A subject of the present invention is also a kit for detecting the immune state of an individual, characterized in that it comprises at least one reagent as defined above, combined with a means for detecting CD4+ T lymphocytes specific for an antigen.

The CD4+ T lymphocytes specific for an antigen are detected by any means known in themselves. For example, use may be made of direct means such as lymphocyte proliferation assays or flow cytometry in the presence of multimeric complexes as defined above, or else indirect means, for instance the assaying of cytokines such as IL2, IL4, IL5 or γIFN, in particular by immunoenzymatic techniques (ELISA, RIA, ELISPOT).

More precisely:

-   -   as regards the proliferation assay:

A suspension of cells (PBMCs, CD8+ cell-depleted PBMCs, T lymphocytes pre-enriched by a step consisting in culturing in vitro with the peptides as defined above, or cloned T lymphocytes) is cultured for 3 to 5 days in the presence of said HLA-DP4 ligands and, as needed, of appropriate presenting cells, such as dendritic cells, autologous or heterologous PBMCs, lymphoblastoid cells such as those obtained after infection with the EBV virus or genetically modified cells. The proliferation of the cells is measured by incorporation of tritiated thymidine into the DNA of the cells. The peptides as defined above make it possible to detect in the initial suspension the presence of cells specific for these peptides.

-   -   as regards the ELISPOT assay:

The ELISPOT assay makes it possible to detect the presence of γIFN-secreting T cells specific for a peptide as defined above.

More precisely, the T cells are detected by measuring the secretion of γIFN after incubation of the PBMCs from patients with said peptides in accordance with the method described in International Application WO 99/51630 or Gahéry-Ségard et al., (J. Virol., 2000, 74, 1964).

-   -   as regards the use of multimeric complexes and in particular of         tetrameric complexes:         -   a biological sample, preferably peripheral blood mononuclear             cells (PBMCs), is brought into contact with tetrameric             complexes as defined above, and         -   labeled cells are analyzed by flow cytometry.

Advantageously, prior to bringing the biological sample into contact with said complex, it is enriched in CD4+ T cells by bringing it into contact with anti-CD4 antibodies so as to enrich said sample.

The tetramers are prepared as specified, for example, in NOVAK et al. (J. Clin. Investig., 1999, 104, R63-R67) or in KURODA et al. (J. Virol., 2000, 74, 18, 8751-8756).

Briefly, the tetramers are produced by incubating, for 72 hours at 37° C. and in a 10 mM citrate phosphate buffer containing 0.15 M NaCl, at a pH of between 4.5 and 7, soluble and biotinylated HLA II molecules with a 10-fold excess of HLA-DP4 ligands as defined above.

The tetramerized form is obtained by adding streptavidin labeled with a fluorochrome to the preparation, in an amount four times less (mole for mole) than the amount of HLA II molecules. The entire mixture is incubated overnight at ambient temperature.

To use these tetramers, a suspension of cells (PBMCs, CD8+ cell-depleted PBMCs, T lymphocytes pre-enriched by a step consisting in culturing in vitro with the HLA-DP4 ligands as defined above, or cloned T lymphocytes) is brought into contact with one or more tetramers (10 to 20 mg/ml) for 1 to 3 hours. After washing, the suspension is analyzed by flow cytometry: the labeling of the cells with the tetramers is visualized by virtue of the fact that these constructs are fluorescent.

The flow cytometry makes it possible to separate the cells labeled with the tetramers from the nonlabeled cells and to thus perform cell sorting.

A subject of the present invention is thus also a method of sorting CD4+ T lymphocytes specific for an antigen, characterized in that it comprises at least the following steps:

-   -   bringing a cell sample into contact with tetramers labeled with         a fluorochrome, prepared from complexes between HLA-DP4 ligands         as defined above and soluble HLA-DP4 molecules, and     -   sorting these cells bound to said tetramers, by flow cytometry.

Besides the above arrangements, the invention also comprises other arrangements, which will emerge from the following description which refers to examples of implementation of the subject of the present invention, with references to the attached drawings in which:

-   -   FIG. 1 illustrates the activity of binding of peptides to the         HLA-DP4 molecules encoded, respectively, by DPB1*0401 (A) and         DPB1*0402 (B), determined according to the method in accordance         with the invention with, as tracer peptide, the biotinylated         UNK1 peptide (10 nM); the percentage of DP4 molecule-binding is         expressed as a function of the molar concentration of the         peptides. The maximum binding (100%) corresponds to the value         obtained for the tracer peptide alone, in the absence of         competitor peptide. UNK: UNK1 (SEQ ID NO:14), IL: IL3 127-146         (SEQ ID NO:13), MAG: MAG 245-258 (SEQ ID NO:19), NSP2: SEQ ID         NO:4, TT: TT 947-963 (SEQ ID NO:20), DQB: DQB 43-57 (SEQ ID         NO:23), HCI 46-63: SEQ ID NO:24) and HA: HA 306-318 (SEQ ID         NO:21),     -   FIG. 2 illustrates the correlation between the HLA-DP4-binding         score (estimated by the method of identifying HLA-DP4-ligand         peptides in accordance with the invention) and the affinity for         the HLA-DP4 molecules (determined by the IC₅₀ value, measured         using the HLA-DP4-binding assay defined in the method of         selecting HLA-DP4-ligand peptides in accordance with the         invention) analyzed on a set of 44 peptides.

EXAMPLE 1 Principle of the HLA-DP4/Peptide Binding Assay 1) Peptide Preparation

All the peptides were synthesized according to the Fmoc strategy in parallel solid-phase synthesis, purified by HPLC and controlled by mass spectrometry (ES-MS).

The peptides are biotinylated on their NH₂-terminal residue, according to the protocol as described in Texier et al., mentioned above.

2) Antibody Preparation

The HLA-DP molecule-specific antibodies, such as the antibody B7/21 (WATSON, et al., Nature, 1983, 304, 358-361), are purified from the culture supernatant of the corresponding hybridomas, on protein A-sepharose columns. These antibodies are then coupled onto sepharose 4B or protein A-sepharose columns for purification of the HLA-DP4 molecules.

More precisely, after centrifugation at 1100 g, the culture supernatant of the B7/21 antibody-producing cells is filtered through 0.22 μm and its pH is adjusted to 7-8 with 0.1 M Tris-HCI buffer, pH 8. This supernatant is then applied to a 10 ml protein A-sepharose 4 fast flow column prewashed with 100 ml of 0.1 M Tris-HCI buffer, pH 8. The column is then washed with 100 ml of 0.1 M Tris-HCI buffer, pH 8, and 100 ml of 0.01 M Tris-HCI buffer, pH 8. The antibodies are eluted with 0.1 M glycine-HCI buffer, pH 3. The column is rinsed with 100 ml of 0.1 M Tris-HCI buffer, pH 8. The eluted fraction which contains the B7/21 antibody is immediately neutralized with 1 M Tris-HCI buffer, pH 8, before being thoroughly dialyzed against 0.1 M borate buffer, pH 8.2. The amount of antibodies obtained is determined based on the optical density (OD) at 278 nm.

The affinity columns intended for purification of the HLA-DP4 molecules are prepared in the following way: 0.75 g of protein A-sepharose 4B (3 ml of final gel) is swollen in water and then in 0.1 M borate buffer pH 8.2. 15 mg of monoclonal antibody, such as B7/21, in 0.1 M borate buffer, pH 8.2, are added to the gel, centrifuged beforehand. The coupling is carried out for two hours at ambient temperature and then controlled by absorbence of the supernatant at 278 nm. The gel is then washed successively with 100 ml of 0.1 M borate buffer, pH 8.2, 120 ml of 0.2 M triethanolamine buffer, pH 8.2, 120 ml of 20 mM dimethylpyrimidate buffer, 0.2 M triethanolamine, pH 8.2 and 150 ml of 0.2 M ethanolamine buffer, pH 8.2. After the gel has been poured, it is rinsed with 150 ml of 0.1 M borate buffer, pH 8.2. The final control for the coupling is performed by an elution in 0.1 M glycine buffer, pH 2.5, containing 0.5 M NaCl; the absorbence at 278 nm of the 1 ml fractions should be less than 0.1. The column is immediately rinsed with 50 ml of 0.1 M borate buffer, pH 8.2, and 20 ml of 0.1 M borate buffer containing 0.02% NaN₃, pH 8.2. It is stored in this buffer at 4° C. until use.

3) Purification of the HLA-DP4 Molecules

The HLA-DP4 molecules are purified from various human B lymphocyte lines transformed with the Epstein Barr virus (EBV), which are homozygotes for DP, by immunoaffinity using monoclonal antibodies specific for all DP molecules. The origin of the lines and the alleles which characterize them are indicated in table IV.

TABLE IV DP DPA1 DPB1 Lines specificity allele allele Reference HOM2 DP4 DPA1*0103 DPB1*0401

BOLETH DP4 DPA1*0103 DPB1*0401

PITOUT DP4 DPA1*0103 DPB1*0401 SOUTHWOOD et al., mentioned above HHKB DP4 DPA1*0103 DPB1*0401 DAVENPORTH et al., P.N.A.S., 1995, 92, 6567 SHU DP4 DPA1*0103 DPB1*0402

MLF DP4 DPA1*0103 DPB1*0402

BM92 DP4 DPA1*0103 DPB1*0402

 the origin of the lines is described on the internet site of the European cell culture collection (http://fuseiv.co.uk/camr/).

The HLA-DP4 molecules are purified from a pellet of these EBV-transformed human cells, according to a protocol derived from those used for the HLA-DR molecules (GORGA et al., J. Biol. Chem., 1987, 262, 16087; TEXIER et al., mentioned above).

More precisely, 5 to 6×10⁹ cells are lysed at a concentration of approximately 10⁸ cells/ml in lysis buffer (0.01 M Tris, 0.15 M NaCl, 0.02% NaN₃, pH 7, 1% NP40, 10 μg/ml aprotinin, 5 mM EDTA, 10 μM PMSF) in ice for 30 minutes. The large cell debris is removed from the lysis medium by centrifugation at 1100 g for 10 minutes at 4° C. The supernatant is then ultracentrifuged at 100 000 g at 4° C. for 1 hour. The rest of the purification takes place in a cold room at 4° C. The lysate is passed successively over a sepharose 4B column (10 ml of gel prepared in 1×PBS), a protein A-sepharose 4B column (5 ml of gel prepared in 1×²PBS) and then over the anti-DP affinity column. The columns are then rinsed with 250 ml of lysis buffer. The sepharose 4B column is discarded. The protein A-sepharose 4B column is rinsed with 25 ml of 1×TBS buffer (0.01 M Tris, 0.15 M NaCl, 0.02% NaN₃, pH 7), 50 ml of (0.1 M glycine; 0.5 M NaCl, pH 2.5) buffer and 200 ml of 1×PBS buffer, before being stored at 4° C. The anti-DP column is rinsed with 250 ml of TBS buffer containing 1 mM of dodecyl maltoside (DM). It is then eluted individually with the elution buffer (100 mM Na₂CO₃, 500 mM NaCl, 0.02% NaN₃, 1.1 mM DM, pH 11.5) in 15 fractions of 3 ml. The eluate is immediately neutralized with 10% of buffer (2 M Tris-HCI, pH 6.8), and then thoroughly dialyzed at 4° C. against 1×PBS buffer containing 1 mM of DM.

4) HLA-DP4-Peptide Binding Assay

The assay for binding of the peptides to the HLA-DP4 molecules is a competition assay with immunoenzymatic detection, derived from that developed for HLA-DR molecules (HLA-DR1: MARSHALL et al., mentioned above), HLA-DR1, -DR2, -DR3, -DR4, -DR7, -DR11 and -DR13: patent FR 99 0879 and TEXIER et al., mentioned above). It is carried out in 96-well plates, which makes it possible to study many samples in the same experiment. Briefly, the purified HLA-DP4 molecules are incubated with a biotinylated peptide which serves as a tracer, and various concentrations of the test peptide. The biotinylated peptide is a DP4-ligand peptide; it is a peptide recognized by DP4-specific CD4+ T lymphocytes, such as those specified in table III above, or a new peptide isolated using the present DP4-binding assay. Among these peptides, mention may be made, for example, of the UNK1 peptide or the IL3 127-146 peptide. The incubation is carried out in a buffer, the pH of which can vary. It is generally 5, and the incubation generally lasts 24 h. After incubation, the samples are neutralized, and then 100 μl of each sample is transferred onto an ELISA plate pre-coated with an anti-DP antibody, such as B7/21. The HLA-DP molecule/biotinylated peptide complexes attached to the bottom of the plate via the antibody are revealed by means of streptavidin phosphatase and a fluorescent substrate. The activity of each peptide is characterized by the concentration which inhibits 50% of the binding of the biotinylated peptide (IC₅₀).

EXAMPLE 2 Determination of the Binding Assay Parameters 1) Tracer Peptide a) Materials and Methods

The binding of peptides to the two DP4 molecules (DP401 encoded by the DPA1*0103/DPB1*0401 allele and DP402 encoded by the DPA1*0103/DPB1*0402 allele) was analyzed by ELISA using the following direct binding assay:

The HLA-DP4 molecules purified according to the protocol described in example 1 are diluted 10 times in 10 mM phosphate buffer (1/10 dilution). They are then incubated with various concentrations of a biotinylated peptide (10⁻⁶ M, 10⁻⁷ M and 10⁻⁸ M) in 10 mM phosphate buffer containing 150 mM NaCl, 1 mM DM, 10 mM citrate and 0.003% thimerosal, pH 5, in 96-well polypropylene plates, for 24 h at 37° C. Samples without DP4 molecules are used as a control. At the end of the incubation, the samples are neutralized with 50 μl of 450 mM Tris-HCI buffer, pH 7.5, containing 0.003% thimerosal, 0.3% BSA and 1 mM DM. They are then transferred onto 96-well maxisorp ELISA plates onto which the anti-DP antibodies have been pre-adsorbed. Specifically, 10 μg/ml of anti-DP antibodies were incubated overnight at 4° C. (100 μl/well) and the plates were then saturated with the 100 mM Tris-HCI buffer, pH 7.5, containing 0.3% BSA and 0.003% thimerosal overnight at 4° C. The incubation of the samples on these plates is carried out for two hours at ambient temperature, like the remainder of the assay, and then extensive washing is performed, between each step, in 0.1 M Tris-HCI buffer, pH 7.5, containing 0.05% Tween-20. The biotinylated peptide bound to the HLA-DP molecules is detected by adding 100 μl/well of the streptavidin-alkaline phosphatase conjugate (45 minutes) diluted 1/2000 in the 10 mM Tris buffer, pH 7, containing 0.15 M NaCl, 0.05% Tween 20, 0.2% BSA and 0.003% thimerosal, and then by adding 200 μl/well of 100 μM MUP substrate in 0.05 M NaHCO₃ buffer, pH 9.8, containing 1 mM MgCl₂. The emission of fluorescence by the product of the enzymatic reaction is measured at 450 nm after excitation at 365 nm, and the ratio of the values obtained with or without DP4 is determined (R_(F)=fluorescence value in the presence of DP4/fluorescence value in the absence of DP4).

b) Results

The DP401 molecule-binding of peptides derived from the DP4-ligand peptides described above (table III) was assayed:

-   -   peptide bUL21 283-302 (RELWWVFYAGDRALEEPHAE; SEQ ID NO: 18)     -   peptide bIL3 127-146 (SEQ ID NO:13)     -   peptide bMAG 245-258 (LLTQHFVQENYLEY; SEQ ID NO:19)     -   peptide bMT 451-466 (SEQ ID NO:6)     -   peptide bNS-p2 (SEQ ID NO:4)     -   peptide bTT 947-963 (FNNFTVSFWLRVPKVSA; SEQ ID NO:20)     -   peptide bUNK1 (SEQ ID NO:14)

Two peptides specific, respectively, for DR1 and for DR7 were used as control for the DP4-binding specificity:

-   -   peptide bHA 306-318 (PKYVKQNTLKLAT; SEQ ID NO:21) described by         HILL et al., J. Immunol., 1994, 152, 2890, and     -   peptide bYKL (AAYAAAKAAALAA; SEQ ID NO:22) described by MARSHALL         et al., mentioned above.

The results are given in table V.

TABLE V Selection of the tracers using a direct HLA- DP4-binding assay R_(F) = fluorescence value in the presence of DP4/ fluorescence value in the absence of DP4 peptide 10⁻⁶M 10⁻⁷M 10⁻⁸M bUL21 283-302 8.3 13.3 10.1 bIL3 127-146 10 20 17.3 bMAG 245-258 1.5 6.7 5.4 bMT 451-466 5.5 4 2 bNS-p2 1 4.2 7.8 bTT 947-963 1.7 3.6 2.7 bUNK1 21.6 21.7 20 bHA 306-318 4.9 4.7 2.7 bYKL 4.8 3.2 3

The results show that:

-   -   the bUNK1 and bIL3 127-146 peptides have a high DP4-binding         capacity,     -   the bTT 947-963 and bMT 451-466 peptides have a low binding         capacity, and     -   the bNS-p2, pUL21 283-302 and bMAG 245-258 peptides have an         intermediate binding capacity.

The peptides exhibiting an R_(F)>5 at the concentration of 10⁻⁸ M are considered to be good tracers which can be used in the binding assay.

The UNK1 peptide, which gave the best results, was used to optimize the binding assay.

2) Time, pH, Concentration of the Tracer Peptide

In order to have a sensitive and DP4-specific assay, the concentration of HLA-DP4 molecules, the concentration of the biotinylated peptide, the pH and the peptide/HLA-DP4 molecule incubation time were optimized with the bUNK1 peptide.

a) Materials and Methods

The HLA-DP4 molecules purified according to the protocol described in example 1 were diluted 1/10, 1/20, 1/40 and 1/80 in 10 mM phosphate buffer containing 150 mM of NaCl, 1 mM DM, 10 mM citrate and 0.003% thimerosal, at various pH values (pH 4, 5, 5.5, 6, 6.5 and 7), with the bUNK1 peptide at various concentrations and several concentrations of competitor peptides, in 96-well polypropylene plates. At the end of the incubation at 37° C., the samples were neutralized with 50 μl of 450 mM Tris-HCI buffer, pH 7.5, containing 0.003% thimerosal, 0.3% BSA and 1 mM DM. They were then transferred onto 96-well maxisorp ELISA plates onto which the anti-DP antibodies had been pre-adsorbed. Very specifically, 10 μg/ml of anti-DP antibodies were incubated overnight at 4° C. (100 μl/well) and the plates were then saturated with the 100 mM Tris-HCI buffer, pH 7.5, containing 0.3% BSA and 0.003% thimerosal overnight at 4° C. The incubation of the samples on these plates was carried out for two hours at ambient temperature, like the remainder of the assay, and then thorough washing was performed, between each step, in 0.1 M Tris-HCI buffer, pH 7.5, containing 0.05% Tween-20. The biotinylated peptide bound to the HLA-DP molecules was detected by adding 100 μg/well of the streptavidin-alkaline phosphatase conjugate (45 minutes) diluted 1/2000 in the 10 mM Tris buffer, pH 7, containing 0.15 M NaCl, 0.05% Tween 20, 0.2% BSA and 0.003% thimerosal, and then by adding 200 μl/well of 100 μM MUP substrate in 0.05 M NaHCO₃ buffer, pH 9.8, containing 1 mM MgCl₂. The emission of fluorescence by the product of the enzymatic reaction was measured at 450 nm after excitation at 365 nm. The maximum binding was determined by incubating the biotinylated peptide with the MHC II molecule in the absence of competitor peptide. The binding specificity was controlled by adding an excess of nonbiotinylated peptide. The background noise obtained does not differ significantly from that obtained by incubating the biotinylated peptide in the absence of the MHC II molecules.

The results are expressed in the form of the concentration of competitor peptide which inhibits 50% of the maximum binding of the labeled peptide (IC₅₀).

b) Results

The optimum conditions are given in table VI:

TABLE VI Conditions for the DP4 molecule-binding assay DPA1*0103/ DPA1*0103/ Molecules DPB1*0401 DPB1*0402 Biotinylated bUNK 1-17 bUNK 1-17 peptide Concentration 10 nM 10 nM pH 5 5 Incubation 24 h 24 h time Concentration Dilution of HLA-DP4 1/20 to 1/4

he dilutions indicated are carried out using the preparation of purified HLA-DP4 molecules obtained in example 1.

EXAMPLE 3 Sensitivity and Specificity of the DP4 Molecule-Binding Assay a) Specificity

The results illustrated in FIG. 1 show that the binding activity measured in the assay is specific for HLA-DP4 insofar as:

-   -   peptides known to be ligands for DP4 molecules effectively bind         the HLA-DP*0401 and 0402 molecules. They are the IL3 127-146         peptide (naturally present on a DP4 molecule), DP4-restricted         CD4+ T lymphocyte-specific peptides: NS-p2 (or NSP2), TT 947-963         and, to a lesser extent, the MAG 245-258 peptide, and     -   peptides known to bind other HLA II molecules do not bind to the         HLA-DP*0401 and 0402 molecules. They are the peptides: DQB 43-57         (DVEVYRAVTPLGPPD, SEQ ID NO:23), HCI 46-63 (EPRAPWIEQEGPEYWDQE,         SEQ ID NO:24) and HA 306-318 (SEQ ID NO:21) which are known to         bind, respectively, to HLA-DQ3, HLA-DQ2 and HLA-DR molecules         (MARSHALL et al., mentioned above, JOHANSEN et al.,         Immunogenetics, 1996, 45, 142).

The specificity of the assay results from the use:

-   -   of an HLA-DP molecule-specific antibody for the purification and         for the adsorption of the ELISA plates, and     -   of the biotinylated peptide which binds with high affinity; FIG.         1 shows that the UNK peptide, which is the nonbiotinylated         counterpart of the bUNK tracer peptide, completely inhibits the         binding of the tracer both to the HLA-DPB1*0401 molecule and to         the DPB1*0402 molecule.

b) Sensitivity

The sensitivity of the assay is reflected by the IC₅₀ observed with the nonbiotinylated peptide (UNK1) corresponding to the tracer (bUNK1). FIG. 1 indicates that the values of, respectively, 8 and 9 nM for DPB1*0401 and DPB1*0402 reflect good sensitivity.

FIG. 1 also shows that the DPB1*401 molecule-binding and DPB1*402 molecule-binding activities of the peptides, although comparable, are different. These results confirm that the DPB1*401 and DPB1*402 molecules exhibit differences which can be detected by this binding assay.

EXAMPLE 4 Screening for HLA-DP4-Ligand Peptides Using a Peptide Library 1) Materials and Methods

The overlapping peptides carrying the complete sequence of the major allergen of bee venom (Api ml), described in patent FR 99 00879, were synthesized and subjected to the DP401 molecule-binding and DP402 molecule-binding assays, under the conditions defined in table VI.

2) Results

The results are given in table VII below:

TABLE VII Binding of the Api m1 peptides to the HLA- DPB1*0401 and DPB1*0402 molecules IC₅₀ (nM) Peptide DP0401 DP0402  1-18 >10 000 >10 000  5-22 >10 000 >10 000  9-26 >10 000 >10 000 13-30 >10 000 >10 000 17-34 >10 000 >10 000 21-38 >10 000 >10 000 25-42 >10 000 >10 000 29-46 >10 000 >10 000 33-50 >10 000 >10 000 37-54 >10 000 >10 000 41-58 >10 000 >10 000 45-62 >10 000 >10 000 49-66 >10 000 >10 000 53-70 >10 000 >10 000 57-74 >10 000 >10 000 61-78 >10 000 >10 000 65-82   50 000     6500 69-86 >10 000 >10 000 73-90 >10 000 >10 000 77-94     450     175 81-98     2250     1225  85-102 >10 000 >10 000  89-106 >10 000 >10 000  93-110 >10 000 >10 000  97-114 >10 000 >10 000 101-118 >10 000 >10 000 105-122 >10 000 >10 000 109-126 >10 000 >10 000 113-130 >10 000 >10 000 117-134 >10 000 >10 000

These results show that peptide 77-94 of the major antigen of bee venom 94 (Api ml 77-94: TISSYFVGKMYFNLIDTK, SEQ ID NO:17) is a peptide which is a ligand for the DPB1*0401 and DPB1*0402 molecules.

EXAMPLE 5 Determination of the DP4 Molecule-Binding Units 1) Materials and Methods

a) Determination of a Minimum Peptide Derived from UNK1 Capable of Binding to the DP4 Molecules

Peptides derived from the UNK1 peptide (UNK 1-17, SEQ ID NO:14) comprising increasing deletions at one of the NH₂ or COOH ends, or at both ends, were synthesized. The sequence of these peptides (UNK 1-11, 1-12, 1-13, 2-14, 3-15, 4-16, 5-17, 6-17, 7-17, 3-17 and 1-15) corresponding, respectively, to SEQ ID NOS:25-26, 16 and 27-34 is given in table VIII. The binding activity of the peptides, expressed by the IC₅₀ value, was determined using the competition binding assay, under the conditions defined in table VI.

b) Determination of the Residues of the UNK1 Peptide which are Involved in Binding to the DP4 Molecules

Mutants of the UNK 3-15 peptide (KYFAATQFEPLAA; SEQ ID NO:28) were synthesized; each mutant contains only one of the residues Y4, F5, T8, Q9, F10, E11, P12 and L13 substituted with alanine or with lysine, the residue K3 substituted with alanine, or else one of the residues A6, A7, A14 and A15 substituted with lysine.

The sequence of these peptides (UNK K3A, Y4A, FSA, TBA, Q9A, F10A, E11A, P12A, L13A, Y4K, FSK, A6K, A7K, T8K, Q9K, F10K, E11K, P12K, L13K, A14K and A15K) corresponding, respectively, to the sequences SEQ ID NOS:35 to 55 is given in table IX.

The binding activity of the peptides, expressed by the IC₅₀ value, was determined using the competition binding assay, under the conditions defined in table VI. The loss of binding of the mutant peptides is expressed by the ratio of the IC₅₀ values for the mutant peptide and for the UNK1 peptide.

c) Determination of the DP401 Molecule-Binding and DP402 Molecule-Binding Units

The residues F in P1, T in P4, F in P6 or L in P9 of the UNK1 peptide were substituted with one of the following residues:

-   -   P1: Y, L, E, N, T, D, G, H, I, M, P, Q, R, S, V or W,     -   P4: F, L, E, D, N, Y, R, S, G, H or P     -   P6: Y, L, W, E, N, T, D, G, H, I, M, P, Q, R, S, V, W or C,     -   P9: F, Y, E, D, N, R, V, G, H, I, P, Q, S, T or W.

The sequence of these peptides, corresponding to the sequences SEQ ID NOS: 56 to 81 and SEQ ID NOS:96 to 129, is given, respectively, in tables Xa and Xb.

The loss of DP4 molecule-binding of the mutant peptides was determined using the assay described for the alanine and lysine mutants.

2) Results a) Determination of a Minimum UNK1 Peptide

The results are given in table VIII below:

TABLE VIII Binding to DP401 and DP402 of the peptides derived from UNK1 (UNK 1-17) Position of the sequences IC₅₀ (nM) Peptides 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 401 402 UNK1-17 E K K Y F A A T Q F E P L A A R L 11 14 UNK1-11 E K K Y F A A T Q F E 55 95 UNK1-12 E K K Y F A A T Q F E P 93 100 UNK1-13 E K K Y F A A T Q F E P L 14 28 UNK2-14 K K Y F A A T Q F E P L A 15 23 UNK3-15 K Y F A A T Q F E P L A A 19 23 UNK4-16 Y F A A T Q F E P L A A R 38 25 UNK5-17 F A A T Q F E P L A A R L 65 38 UNK6-17 A A T Q F E P L A A R L 15000 6000 UNK7-17 A T Q F E P L A A R L 15000 12500 UNK3-17 K Y F A A T Q F E P L A A R L 18 14 UNK1-15 E K K Y F A A T Q F E P L A A 18 17

The results obtained show that:

-   -   the loss of binding observed with the UNK 1-11, UNK 1-12, UNK         4-16 and UNK 5-17 peptides suggests that the minimum peptide is         13 to 15 amino acids in size,     -   the loss of binding observed with the UNK 6-17 and UNK 7-17         peptides suggests that the F residue at position 5 is the first         residue for anchoring of the peptides in the binding site of the         DP4 molecules (residue P1).         b) Determination of the Residues of the UNK1 Peptide which are         Involved in Binding to the DP4 Molecules

The results are given in table IX below:

TABLE IX Loss of binding to DP401 and DP402 of the alanine and lysine mutants PEPTIDE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 DP*0401 DP*0402 UNK1 E K K Y F A A T Q F E P L A A R L 1 1.00 UNK 3-15 K Y F A A T Q F E P L A A 1.5 1.7 P1 P4 P6 P9 UNK K3A A Y F A A T Q F E P L A A 0.9 1 UNK Y4A K A F A A T Q F E P L A A 1.2 0.9 UNK F5A K Y A A A T Q F E P L A A 41 5.7 UNK T8A K Y F A A A Q F E P L A A 0.8 0.8 UNK Q9A K Y F A A T A F E P L A A 0.8 0.6 UNK F10A K Y F A A T Q A E P L A A 336 62 UNK E11A K Y F A A T Q F A P L A A 0.5 0.5 UNK P12A K Y F A A T Q F E A L A A 1.2 0.9 UNK L13A K Y F A A T Q F E P A A A 3.1 2.3 UNK Y4K K K F A A T Q F E P L A A 1.2 1 UNK F5K K Y K A A T Q F E P L A A 569 297 UNK A6K K Y F K A T Q F E P L A A 1.6 1 UNK A7K K Y F A K T Q F E P L A A 2 1.2 UNK T8K K Y F A A K Q F E P L A A 31 3.6 UNK Q9K K Y F A A T K F E P L A A 1.6 0.9 UNK F10K K Y F A A T Q K E P L A A 420 350 UNK E11K K Y F A A T Q F K P L A A 1.3 0.7 UNK P12K  K Y F A A T Q F E K L A A 1.8 1.4 UNK L13K K Y F A A  T Q F  E P K A A 81 70 UNK A13K K Y F A A T Q F E P  L K A 1.7 1.4 UNK A15K K Y F A A T Q F E P L A K 1.7 1.4

The results show a loss of binding for F5A, F10A, F5K, F10K and L13K, which strongly suggests that the residues for anchoring of the peptides in the binding site of the DP4 molecules are, respectively, F5 at position P1, F10 at position P6 and L13 at position P9.

c) Determination of the DP401 Molecule-Binding and DP402 Molecule-Binding Units

The results are given in tables Xa and Xb and table XI below:

TABLE Xa Loss of binding to DP401 and DP40

 of the mutants at P1, P4, P6 and P9 (SEQ ID NOS: 56 to 81) PEPTIDE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 DP*0401 DP*0402 UNK1 E K K Y F A A T Q  F E P L A A R L 1.00 1.00 UNK 3-15 K Y F A A T Q F E P L A A 1.4 1.4 P1 P4 P6 P9 UNK F5Y K Y Y A A T Q F E P L A A 3 3.9 UNK F5L K Y L A A T Q F E P L A A 3.6 6 UNK F5E K Y E A A T Q F E P L A A 117 123 UNK F5N K Y N A A T Q  F E P L A A 398 76 UNK F5T K Y T A A T Q  F E P L A A 234 126 UNK T8F K Y F A A F Q F E P L A A 2.7 1.7 UNK T8L K Y F A A L Q F E P L A A 1.9 2.6 UNK T8E K Y F A A E Q F E P L A A 2.7 3.7 UNK T8D K Y F A A D Q F E P L A A 2 2.3 UNK T8N K Y F A A N Q F E P L A A 4.1 4.8 UNK T8Y K Y F A A Y Q F E P L A A 4.1 2.4 UNK T8R K Y F A A R Q F E P L A A 17 6.7 UNK T8S K Y F A A S Q F E P L A A 2.7 3.4 UNK F10Y K Y F A A T Q Y E P L A A 2.4 3.4 UNK F10L K Y F A A T Q L E P L A A 7.2 6.7 UNK F10W K Y F A A T Q W E P L A A 2.2 2.5 UNK F10E K Y F  A A T Q  E E P L A A 1405 1190 UNK F10N K Y F  A A T Q  N E P L A A 2460 1809 UNK F10T K Y F  A A T Q T E P L A A 1171 476 UNK L13F K Y F A A T Q F E P F A A 1.7 4 UNK L13Y K Y F A A T Q F E P Y A A 2 15 UNK L13E K Y F A A T Q F E P E A A 11 8 UNK L13D K Y F A A T Q F E P D A A 9.4 9 UNK L13N K Y F A A T Q F E P N A A 17 10 UNK L13R K Y F A A T Q  F E P R A A 64 43 UNK L13V K Y F A A T Q F E P V A A 2.5 1.9

he values greater than 10 are indicated in bold

TABLE Xb Loss of binding to DP401 and DP40

 of the mutants at P1, P4, P6 and P9 (SEQ ID NOS: 96 to 129) Peptides 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Loss UNK E K K Y F A A T Q F E P L A A R L DP*0401 DP*0402 UNK F5D K Y D A A T Q F E P L A A 150 57 UNK F5G K Y G A A T Q F E P L A A 160 55 UNK F5H K Y H A A T Q F E P L A A 150 36 UNK F5I K Y I A A T Q F E P L A A 3 2 UNK F5M K Y M A A T Q F E P L A A 3 3 UNK F5P K Y P A A T Q F E P L A A 100 48 UNK F5Q K Y Q A A T Q F E P L A A 260 97 UNK F5R K Y R A A T Q F E P L A A 140 54 UNK F5S K Y S A A T Q F E P L A A 160 42 UNK F5V K Y V A A T Q F E P L A A 11 6 UNK F5W K Y W A A T Q F E P L A A 0.5 1 UNK T8G K Y F A A G Q F E P L A A 1 2 UNK T8P K Y F A A P Q F E P L A A 3 2 UNK T8H K Y F A A H Q F E P L A A 6 2 UNK F10D K Y F A A T Q D E P L A A 2700 120 UNK F10G K Y F A A T Q G E P L A A >7100 3700 UNK F10H K Y F A A T Q H E P L A A 2400 220 UNK F10I K Y F A A T Q I E P L A A 7 6 UNK F10M K Y F A A T Q M E P L A A 4 1 UNK F10P K Y F A A T Q P E P L A A 210 12 UNK F10Q K Y F A A T Q Q E P L A A >7100 230 UNK F10R K Y F  A A T Q R E P L A A >7100 370 UNK F10S K Y F A A T Q S E P L A A 500 33 UNK F10V K Y F A A T Q  V E P L A A 42 22 UNK F10W K Y F A A T Q W E P L A A 1 1 UNK F10C K Y F A A T Q C E P L A A 17 2 UNK L13G K Y F A A T Q F E P G A A 15 12 UNK L13H K Y F A A T Q F E P H A A 13 35 UNK L13I K Y F A A T Q F E P I A A 1 3 UNK L13P K Y F A A T Q F E P P A A 9 3 UNK L13Q K Y F A A T Q F E P Q A A 8 2 UNK L13S K Y F A A T Q F E P S A A 9 5 UNK L13T K Y F A A T Q F E P T A A 5 3 UNK L13W K Y F A A T Q F E P W A A 1 13

he values greater than 10 are indicated in bold

TABLE XI Specificity of the DP4 molecule bindin

P1 P4 P6 P9 + − + − + − + − DP* F, Y, H, T, F, L, K, R F, Y, H, V, F, Y , H, G, 0401 L, I, S, A, Y, H, L, W, A, G, V, A, K, R, M, W G, P, T, A, I, M T, P, I, P, E, N, C K, R, S, G, S, K, W , L, D, E, P, I, R, E, M, Q, N, Q, M, Q, D, N, S, T, C, V E, D, Q, C D N, V, W, C DP* F, Y, H, T, F, L, F, Y, H, V, F, V, Y, H, 0402 L, A , S, G, Y, H, L, W, A, G, A, I, W, G, I, M, P, K, T, A, I, M, C T, P, P, L, K, R, N V , W R, D, S, G, S, K, M, C , E, N, P, I, R, E, Q, S, Q, C MQ, D, N, T, D, E, D, Q E N, V, K, W, C, R

he amino acids which caused, respectively, a loss of binding by a factor greater than 10 and less than 10 are indicated in the column (−) and the column (+); the anchoring residues are represented in bold and the differences between DPB1*0401 and DPB1*0402 are underlined.

The results obtained show that:

-   -   pockets P1, P6 and P9 of the binding site of the DP4 molecules         accept aromatic or hydrophobic residues,     -   pocket P4 of the binding site of the DP4 molecules is very         permissive, as is, to a lesser extent, pocket P9, which also         accepts Q, S, T and D residues, and     -   the DPB1*0401 allele accepts a tyrosine or tryptophan residue at         P9 but does not accept cysteine or glutamic acid residues at         this position, nor alanine or valine residues at P1, nor lysine         or arginine residues at P4, nor a cysteine residue at P6; the         DPB1*0402 allele accepts an alanine or valine residue at P1, a         lysine or arginine residue at P4, a cysteine residue at P6 and a         cysteine or glutamic acid residue at P9, but does not accept a         tyrosine or tryptophan residue at this position.

d) Identification of a Binding Unit in the Sequence of DP4-Ligand Peptides

The binding activity of various DP4-ligand peptides was measured using the competition binding assay, under the conditions defined in table VI. The results are expressed by the IC₅₀ values (table XIII) or by the value of the ratio of the IC₅₀ values for the ligand peptide and for the UNK1 peptide (table XII).

The peptides tested, corresponding respectively to the following sequence SEQ ID NOS, are given in tables XII and XIII:

-   -   TT 947-963 (SEQ ID NO:20)     -   UNK1 9 (SEQ ID NO:14)     -   NY-ESO1 87-11, 119-143, 158-180, 166-180, (SEQ ID NO:94, 83, 82,         95),     -   UL21 283-302 (SEQ ID NO:18)     -   IL3 127-146 (SEQ ID NO:13)     -   NS-P2 (SEQ ID NO:4)     -   Api-ml 65-72, 69-86, 73-90, 77-94, 81-98 (SEQ ID NO: 85, 88, 89,         17, 86)     -   MAG 245-258 (SEQ ID NO:19)     -   MART1 1-20, 41-60, 51-73, 62-72, 103-118 (SEQ ID NO:90, 91, 87,         92, 93).

In parallel, the sequences of these DP4-ligand peptides were aligned and the presence of a DP4-binding unit as defined in table XI was sought (tables XII and XIII).

TABLE XII Alignment of the sequences of the DP4-ligand peptides Peptides Sequences 040

040

P1 P6 P9 TT947-963 FNN F TVSF W LRVPKVSA 0.57 0.75 UNK1 EKKY F AATQ F EPLAARL 1 1 NY-ESO1 LLM W ITQC F LPVFLAQPPSGQRR 2 7 158-180 UL21 283-302 RE L WWVF Y AG D RALEEPHAE 2.86 2.78 IL3 127-146 GPGAPAD V QYDL Y LN V ANRR 3.98 3.06 NS-p2 GVQIVRQIRSGERF L KI W SQ 4.69 3.89 NY-ESO1 PGVLLKEFTVSGN I LTIR L TA A DHR 12 12 119-143 NY-ESO1 PGVL L KEFT V SGNILTIRLTAADHR 12 12 119-143 NY-ESO1 PG V LLKE F TV S GNILTIRLTAADHR 12 12 119-143 NY-ES01 LLEF Y LAMP F ATPMEAELARRSLAQ 20 9 87-111 Api m1 77-94 TISSY F VGKM Y FN L IDTK 30 10 Api m1 77-94 TISSYF V GKMY F NL I DTK 30 10 Api m1 65-72 DKFYDCLKNSADTSSYF NT 371.4 MAG 245-258 L L TQH F VQEN Y LE Y 30.2 38.89 MAG 245-258 L L TQHF V QENYLEY 30.2 38.89 Api m1 81-98 YFVGKMYFNLIDTKCYKL 150 70 MART1 51-73 RNGYRA L MDKS L HVGTQCALTRR 857 414

401: relative DPB1*0401-binding activity of the peptides compared to the UNK1 peptide (binding activity equal to 1)

402: relative DPB1*0402-binding activity of the peptides compared to the UNK1 peptide (binding activity equal to 1)

TABLE XIII Binding of the Api m1, NY-ESO1 and MART1 peptides to DPB1*0401 and DPB*040

IC50 (nM) peptides Sequences 401 402 UNK1 EKKY F AATQ F EP L AARL 12 13 Api m1 65-72 DKFYDCLKNSADTISSYF 50000 6500 Api m1 69-86 DCLKNSADTISSYFVGKM >10000 >10000 Api m1 73-90 NSADTISSY F VGKM Y FN L >10000 >10000 Api m1 77-94 TISSY FV GKMYFNLIDTK 450 175 Api m1 81-98 Y FV GKM YF N LI DTKCYKL 2250 1225 MART1 1-20 MPREDAHFIYGYPKKGHGHS >10000 >10000 MART1 41-60 LLIGCWYCRRRNGYRALMDK >10000 >10000 MART1 51-7

RRNGYRALMDKSLHVGTQCA 8000 4000 LTRR MART1 62-72 LMDKSLHVGTQCALTRRCPQ >10000 >10000 MART1 103-118 AYEKLSAEQSPPPYSP >10000 >10000 NY-ESO1 LLEFYLAMPFATPMEAELAR 183 83 87-111 RSLAQ NY-ESO1 PGVLLKEFTVSGN I LTIR L T 110 117 119-14

A A DHR NY-ESO1 LLM W ITQC F LP V FLAQPPSGQRR 20 67 158-180 NY-ESO1 FLPVFLAQPPSGQRR >10000 >10000 166-180

he amino acids compatible with the unit for binding to the DPB1*0401 and DPB1*0402 alleles are indicated in bold and the complete binding units are underlined.

he sequence of this peptide is described in Zarour et al., PNAS, 2000, 97, 400-405.

he sequence of this peptide is described in Zarour et al., Cancer Res., 2000, 60, 4646-4952.

The results show that, for most of the DP4-ligand peptides, a strong correlation exists between the presence of at least two residues P1 and P6 or P6 and P9 as defined in table XI and a high affinity for the DP4 molecules (IC₅₀<1000 nM). These results also show that the most important residues in the binding to HLA-DP4 are the aromatic or hydrophobic residues at position P1 and P6; these same residues at position P9 are less important.

EXAMPLE 6 Prediction of the Sequence of HLA-DP4-Ligand Peptides Based on an Amino Acid Sequence 1) Materials and Methods

An HLA-DP4 molecule-binding matrix was established based on the binding activities (IC₅₀) of the mutants of the UNK 3-15 peptide, measured using the assay for binding to the DP4 molecules encoded by the DPB1*0401 and DPB1*0402 alleles, as defined above, using the UNK 3-15 peptide as tracer peptide (example 5 and tables IX, Xa, Xb and XI). The contribution of each of the amino acids at positions P1, P4, P6 and P9 of said mutant peptides to the binding to the DP*0401 and DP*0402 molecules is evaluated by a binding score corresponding to the logarithm (log) of the ratio of the IC₅₀ values for the mutant peptide and for the UNK 3-15 peptide. The score for binding to the DP*0401 and DP*0402 molecules, obtained for each of the amino acids at positions P1, P4, P6 and P9 of said mutant peptides, constitutes the HLA-DP4-binding matrix shown in table XIV below:

TABLE XIV HLA-DP4-binding matrix Amino acid DP4 Position A D E F G H I K L M DP*0401 P1 1.6 1.7 1.85 0 2.2 2.17 0.48 2.6 0.48 0.48 P4 0 0.3 X0.3 0.3 0 0.78 0.3 1.48 0.3 0.3 P6 2.38 3.32 2.9 0 3.85 3.4 0.85 2.43 0.78 0.6 P9 0.6 0.85 1.04 0 1.18 1.11 0 1.85 0 0 DP*0402 P1 0.85 1.76 1.85 0 1.76 1.56 0.3 2.28 0.48 0.48 P4 0 0.3 0.48 0 0 0.3 0.3 0.7 0.3 0.3 P6 1.88 3.85 2.9 0 3.57 2.33 0.78 2.28 0.7 0 P9 0.6 0.9 0.9 0.7 1.08 1.54 0.48 1.9 0 0.3 Amino acid DP4 Position N P Q R S T V W Y C DP*0401 P1 2.32 1.98 2.41 2.14 2.2 2.15 1.04 0 0.48 2.32 P4 0.6 0.48 0.3 1.5 0.3 0 0.15 0.3 0.6 0.6 P6 3.15 2.32 3.85 3.85 2.7 2.85 1.62 0.3 0.3 1.23 P9 1.15 0.95 0.9 1.7 0.95 0.6 0.3 0 0.3 1.15 DP*0402 P1 1.78 1.68 1.99 1.72 1.62 1.85 0.78 0 0.48 1.78 P4 0.6 0.3 0.3 0.7 0.48 0 0.15 0 0 0.6 P6 3.08 1.08 2.36 2.57 1.52 2.48 1.34 0.3 0.48 0.3 P9 0.85 0.48 0.3 1.6 0.7 0.6 0.3 1.11 1.18 0.85

Starting with an amino acid sequence, the binding of the peptides of at least 9 amino acids included in said sequence is calculated, based on the matrix above, by adding, for each fragment of 9 amino acids of said peptide, for example overlapping fragments of 9 amino acids covering this entire sequence, the binding scores for the residues at positions 1, 4, 6 and 9 of said fragment.

Peptides having binding scores of, respectively, 0, 1, 2, 3 and 4 correspond to peptides which exhibit a 1-, 10-, 100-, 1000- and 10 000-fold loss of binding relative to the UNK 3-15 peptide (IC₅₀ 10 nM), i.e. exhibiting an estimated binding activity of, respectively, 10 nM, 100 nM, 1000 nM, 10 μM and 100 μM.

The peptides having the lowest binding scores, preferably less than 2, preferably less than 1, even more preferably close to 0, are selected; these peptides correspond to those for which the HLA-DP4-binding activity, estimated based on the binding matrix as defined above, is the highest.

2) Results

The correlation between the binding score for the peptides (estimated using the HLA-DP4-binding matrix, table XIV), and their affinity for the HLA-DP4 molecules (residues in the binding assay as defined above) was analyzed for 44 peptides studied in examples 4 and 5.

The results are given in table XV and FIG. 2.

TABLE XV Binding score and HLA-DP4-binding activity of various peptides DP*0401 DP*0402 Binding Binding Peptide IC₅₀ (nM) score IC₅₀ (nM) score TT947-963 4.61 0.90 7 1.08 UNK 1 9.47 0.00 10.00 0.00 NY-ESO1 158-180 30.00 0.60 70.00 0.60 UL21 283-302 22.89 1.77 20.00 2.01 IL3 127-146 30.37 1.94 30.00 1.86 NSP2 40.00 2.44 40.00 2.69 NY-ESO1 119-143 100.00 2.16 100.00 1.90 NY-ESO1 87-11 186.12 1.73 87.00 1.26 MAG 245-258 250.00 0.90 400.00 1.68 MART1 51-73 8000.00 3.76 4000.00 2.45 Api-m1 1-18 10000.00 1.89 10000.00 2.08 Api-m1 5-22 10000.00 4.88 10000.00 3.33 Api-m1 9-26 10000.00 3.58 10000.00 3.33 Api-m1 13-30 10000.00 2.92 10000.00 2.38 Api-m1 17-34 10000.00 2.92 10000.00 2.38 Api-m1 21-38 10000.00 4.08 10000.00 3.40 Api-m1 25-42 10000.00 3.97 10000.00 2.64 Api-m1 29-46 10000.00 2.56 10000.00 1.86 Api-m1 33-50 10000.00 2.56 10000.00 1.86 Api-m1 37-54 10000.00 4.00 10000.00 3.07 Api-m1 41-58 10000.00 4.00 10000.00 3.07 Api-m1 45-62 10000.00 3.23 10000.00 2.45 Api-m1 49-66 10000.00 3.23 10000.00 2.45 Api-m1 53-70 10000.00 3.15 10000.00 2.45 Api-m1 57-74 10000.00 3.15 10000.00 2.90 Api-m1 61-78 10000.00 3.15 10000.00 2.90 Api-m1 65-82 50000.00 3.64 6500.00 2.90 Api-m1 69-86 10000.00 2.70 10000.00 1.94 Api-m1 73-90 10000.00 1.78 10000.00 1.18 Api-m1 77-94 450 1.34 175.00 1.18 Api-m1 81-98 2250 1.34 1225.00 0.78 Api-m1 85-102 10000.00 1.71 10000.00 0.78 Api-m1 89-106 10000.00 1.71 10000.00 0.78 Api-m1 93-110 10000.00 4.27 10000.00 3.02 Api-m1 97-114 10000.00 4.98 10000.00 3.46 Api-m1 101-118 10000.00 3.67 10000.00 3.21 Api-m1 105-122 10000.00 3.65 10000.00 3.21 Api-m1 109-126 10000.00 3.65 10000.00 3.44 Api-m1 113-130 10000.00 3.96 10000.00 3.50 Api-m1 117-134 10000.00 1.86 10000.00 1.98 MART1 1-20 10000.00 3.16 10000.00 2.16 MART1 41-60 10000.00 2.48 10000.00 2.20 MART1 62-72 10000.00 4.00 10000.00 2.46 MART1 103-118 10000.00 4.11 10000.00 2.95

These results show that a strong correlation exists between the binding score and the affinity of the peptides for HLA-DP4. Specifically, taking as activity threshold a binding score <2 and a binding activity IC₅₀<1000 nM:

-   -   80% of the peptides having a binding score <2 for the HLA-DP4         molecules (DP*0401 and DP*0402) exhibit a high affinity for         these molecules (IC₅₀<1000 nM, true positive peptides), and     -   82% and 76% of the peptides having a binding score ≧2 for the         HLA-DP4 molecules (respectively DP*0401 and DP*0402) exhibit a         low affinity for these molecules (IC₅₀≦1000 nM, true negative         peptides).

Consequently, the HLA-DP4-binding matrix can be used to predict the sequence of HLA-DP4-ligand peptides from any amino acid sequence, in particular a sequence representing an antigen of interest. 

1-26. (canceled)
 27. A method of identifying an HLA-DP4-ligand peptide or a modified HLA-DP40-ligand peptide, which comprises at least one of the following steps: (a) establishing an HLA-DP4-binding matrix by calculating for all the mutants of a tracer peptide, representing all the substitutions of the residues at positions 1, 4, 6 or 9 of said tracer peptide with the other 19 natural amino acids, the ratio of the IC₅₀ values for said mutant peptides and for said tracer peptide, wherein said ratios correspond to the concentration of these molecules which inhibit 50% of the binding of the tracer peptide; (b) evaluating the HLA-DP4-binding of peptides of at least 9 amino acids included in said amino acid sequence, by calculating, for each fragment of 9 amino acids of said peptide, the sum of the scores for HLA-DP4-binding of the residues at positions 1, 4, 6 and 9 of said fragment, using the binding matrix established in (a), and/or (c) identifying the HLA-DP4-ligand peptides corresponding to those to which the loss of HLA-DP4-binding, relative to said tracer peptide, is smallest, i.e. those for which the sum of the binding scores has the lowest value expressed as a logarithm (log); wherein said tracer peptide is at least one peptide selected from the group of consisting of the peptides exhibiting a signal/background noise ratio greater than 5, at a concentration of 10 nM, in a direct HLA-DP4-binding assay, and corresponding to a formula (I) Z₁X₁X₂X₃X₄X₅X₆X₇X₈X₉Z₂; wherein Z₁ and Z₂, which may be identical or different, are zero or each represent a peptide of 1 to 100 natural or synthetic amino acids; X₆ represents an aromatic or hydrophobic amino acid, or a cysteine; X₁ represents an aromatic or hydrophobic amino acid and/or X₉ represents an aromatic or hydrophobic amino acid, or a cysteine (C), an aspartic acid (D), a glutamine (Q), a serine (S), a threonine (T) or a glutamic acid (E); and X₂, X₃, X₄, X₅, X₇ and X₈ each represent a natural or synthetic amino acid.
 28. The method of claim 27, which comprises steps (a), (b), and (c).
 29. The method of claim 27, comprising selecting an HLA-DP4 peptide or modified peptide that exhibits an HLA-DP4-binding activity of less than 1000 nM.
 30. The method of claim 27, which identifies an HLA-DP4 ligand peptide.
 31. The method of claim 27, which identities a modified HLA-DP4 ligand peptide.
 32. The method of claim 27, wherein the HLA-DP4 peptide or modified peptide sequence corresponds to formula (I) Z₁X₁X₂X₃X₄X₅X₆X₇X₈X₉Z₂, wherein Z₁ and Z₂, which may be identical or different, are zero or each represent a peptide of 1 to 100 natural or synthetic amino acids; X₆ represents an aromatic or hydrophobic amino acid, or a cysteine; X₁ represents an aromatic or hydrophobic amino acid; and/or X₉ represents an aromatic or hydrophobic amino acid, or else a cysteine (C), an aspartic acid (D), a glutamine (Q), a serine (S), a threonine (T) or a glutamic acid (E); and X₂, X₃, X₄, X₅, X₇ and X₈ each represent a natural or synthetic amino acid, on condition that said HLA-DP4-ligand peptide of general formula (I) does not correspond to any of the sequences SEQ ID NOS: 1 to
 17. 33. The method of claim 32, wherein X₆ is selected from the group consisting of L, I, W, F, M, Y and C, and X₁ is selected from the group consisting of A, V, L, I, W, F, M and Y; and/or X₉ is selected from the group consisting of A, V, L, I, P, W, F, M, Y, C, D, Q, S, T and E.
 34. The method of claim 33, wherein the peptide or modified peptide hinds specifically to the HLA-DPB1*0401 molecule and X₆ is different from C, and/or X₁ is different from A and from V, and/or X₉ represents W or Y or X₉ is different from E and from C, and/or X₄ is different from K and from R.
 35. The method of claim 33, wherein the peptide or modified peptide hinds specifically to the DPB1*0402 molecule and X₆ represents C, and/or X₁ represents A or V, and/or X₉ represents E or C or X₉ is different from Y and W, and/or X₄ represents K or R. 